A variety of approaches has been used to improve the economy of biologically-based industrial processes by "improving" the organism involved. These techniques constitute what may be categorized as strain improvement programs. The efficacy of improving said processes is dependent on the type of organism and the nature of the end-product.
The success of any strain improvement program will be directly affected by the facility with which genetic diversity can be generated in the subject organism, or alternatively the ease with which the genetic diversity already present in nature can be evaluated.
A colony that appears on agar medium following plating out of spores, cells, or small hyphal fragments consists of a population of cells most of which are genetically identical, although some cells may differ due to spontaneous mutation during the growth of the colony or to nuclear heterogeneity in the original propagule.
It was the rare occurrence of spontaneous mutations within existing cultures that provided the major source of strain improvement germplasm in the early years of the fermentation industry. A secondary source of improved strains was nature itself, that is, the isolation from nature of previously unknown strains with improved characteristics.
As a fuller understanding of the biological and chemical basis of genetic change developed, strain improvement programs incorporated this new knowledge into their rationale. For example, induced mutagenesis to generate genetic diversity followed by the subsequent screening, selection and purification of superior strains represents one of the most effective means of improving the yield of a fermentation product. Mutation programs are vital to the fermentation industry in that higher productivities exhibited by the new strains are essential in reducing costs.
It is now appreciated that the choice of a particular mutagen as well as the actual conditions of mutagenesis can play a major role in determining the types and numbers of mutants recovered during a strain improvement program. In general, two experimental approaches have been used to recover new strains resulting from induced mutagenesis experiments; these are: screening and selection.
In a screening system all strains grow with the exception of those killed outright as a result of the mutagenesis treatment; thus each isolate must be examined to identify the desired characteristic. Since tens of millions of isolates must be examined, this approach can be highly labor intensive.
In a selection system, the experimental conditions are chosen so as to establish a growth differential between the rare strains possessing the desired characteristic and all other strains which do not possess said trait. In certain instances the selected strain will not grow under the conditions of the experiment while the non-selected strains will grow. Thus, by removing the growing strains by filtration or other means, the size of the population of cells remaining to be examined is dramatically reduced. Alternatively, conditions may be established such that the selected strain will grow while the non-selected strains are inhibited, here again effectively reducing the population to be examined.
Although induced mutagenesis has been an extremely powerful force in the area of strain improvement, there are some limitations. For example, as more and more mutations are accumulated in a strain as a result of the continuing improvement program, a saturation level is reached. Subjecting such a strain to further selection often results in a loss of productivity due to reversion of existing mutations.
A more fundamental limitation exists in induced-mutation based improvement programs, namely, such programs are based on the assumption that the strains possess the activity to be improved. In other words, the organism must possess, in its genetic repertoire, the information to direct the synthesis of a gene product before any genetically-based improvement program relating to the function of the product may be considered.
A variety of genetic approaches has been developed to reduce these limitations. For example, hybridization techniques allow for genetic recombination to occur among a number of different strains. Hybridization can be achieved by means of sexual reproduction or asexual processes such as somatic cell fusion or heterokaryon formation. The advent of recombinant DNA technology has reduced the limitations on improvement programs even further. The ability to transfer genes between organisms of widely divergent genetic backgrounds has provided the experimenter with a virtually limitless supply of genetic information upon which to improve. This advent of genetic engineering technology has prompted a renewed interest in natural sources of genetic variability, not with a view toward isolating and developing new strains, per se, but rather as a source of as little as a single gene which may be transferred to already established strains.
Regardless of the source of the variant strain, be it either nature, a spontaneous mutation, an induced mutation, or a recombinant resulting from sexual, asexual or genetic engineering processes, methods of screening and selection remain of critical importance, allowing the experimenter to recover the variant strain from among the population of existing strains from which it arose.
In light of the subject invention, one group of organisms of particular interest with regard to strain improvement programs are those useful for the isomerization of glucose to fructose.
Most food grade glucose is provided as an enzymatic hydrolysate of corn starch, i.e., the corn syrup of commerce. Glucose is generally rated at being 60 to 80% as sweet as sucrose and therefore sells at a correspondingly lower price. It has long been known to isomerize glucose to fructose which is even sweeter than sucrose, by employing an enzyme having glucose isomerase activity. Preferably, such an enzyme is one which has been immobilized onto insoluble supports, such as by crosslinking the enzyme with the support matrix or entrapment in a polymer matrix support such as diethylaminoethyl cellulose or porous glass. The isomerization of glucose provides an equilibrium mixture typically containing 42-50% fructose and is referred to as high fructose corn syrup (HFCS).
Recently, it has been proposed to achieve substantially complete conversion of glucose to fructose by first enzymatically converting glucose to glucosone and thereafter chemically reducing the glucosone to fructose. Thus, in accordance with U.S. Pat. No. 4,246,347, at least about ninety-five percent of D-glucose in aqueous solution is enzymatically oxidized to D-glucosone employing an enzyme having glucose-2-oxidase activity, preferably one obtained from Polyporus obtusus or Aspergillus oryzae, while removing or utilizing co-produced hydrogen peroxide, the D-glucosone being thereafter hydrogenated to D-fructose. As is known in the art, the glucose-2-oxidase obtained from Polyporus obtusus, the preferred organism up to the present, is employed in the form of cell-free extract, primarily because only low enzyme activity is obtained when mycelia of this organism are used as the source of the enzyme.
These conversions, D-glucose to D-glucosone and D-glucosone to D-fructose, can be regarded as proceeding in accordance with the following equations: ##STR1##
Recently, it has been disclosed in U.S. Pat. Nos. 4,442,207 issued Apr. 10, 1984 and 4,447,531 issued May 8, 1984 that various species of Basidiomycetes produce significant quantities of glucose isomerase and glucose-2-oxidase. These findings, particularly when taken in light of the methods of fructose production as described above, warranted the development of a large scale, efficient screening system for the recovery of glucose-2-oxidase producing strains. It is the principle object of the instant invention to provide such a screening system.